After treatment, cells were trypsinized, harvested, and fixed in 1 ml 80 % cold ethanol in test tubes and incubated at 4 °C for 15 min. Cells were then centrifuged at 1,500 rpm for 5 min and the cell pellets were resuspended in 500 μl of PI/RNase staining buffer (BD Biosciences, USA), incubated on ice for 30 min and washed twice with cold PBS. Cell cycle distribution was calculated from 10,000 cells with ModFit LTTM software (Becton Dickinson, CA, USA) using FACS caliber (Becton Dickinson, CA, USA).
Modfit lttm software
Manufactured by BD
Sourced in United States
ModFit LTTM is a software application used for the analysis and modeling of flow cytometry data. It provides tools for cell cycle analysis, DNA content analysis, and other flow cytometry-related data processing and visualization tasks. The software is designed to assist researchers and scientists in the interpretation and understanding of their flow cytometry experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (5 mentions)
Facs caliber, by BD (3 mentions)
Pi rnase staining buffer, by BD (1 mentions)
Dnase free rnase, by Merck Group (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
7 aad, by BioLegend (1 mentions)
Cell cycle and apoptosis analysis kit, by Beyotime (1 mentions)
Accuri c6, by BD (1 mentions)
Mitosox red dye, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
9 protocols using modfit lttm software
1
Cell Cycle Analysis by Flow Cytometry
2
Cell Cycle Analysis by Flow Cytometry
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The cell layers under investigation were harvested and centrifuged at 800× g for 5 min. The cell pellets were washed in phosphate buffered saline (PBS) and then permeabilized with a hypotonic solution containing 10 mM sodium citrate, 2 mM propidium iodide, 20 mM DNase-free RNase (Sigma), and 1% NP-40. The cells were incubated at room temperature for 30 min in the dark and then analyzed using FACScalibur (Becton Dickinson, CA, USA). Cell cycle distribution was calculated from 30.000-40.000 events with ModFit LTTM software (Becton Dickinson) [49 (link)].
3
Cell Cycle Analysis by FACS and Western Blot
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Cell cycle distribution was studied by propidium iodide staining and flow cytometric (FACS) analysis using a FACScalibur (Becton Dickinson, CA, USA) as described in [25 (link)]. The obtained results were calculated from 40.000–60.000 events with ModFit LTTM software (Becton Dickinson, CA, USA). Total cellular extracts were prepared as in [26 (link)]. Nuclear and cytosol extracts were obtained using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA) and analyzed by western blotting as previously described [23 (link), 24 (link)]. Immunoblotting signals were quantified by the analysis of scanner band intensities using ImageJ64 software. To determine the effect of BuA on p27Kip1 half-life, control and 16-hour BuA-treated cells were grown in presence of the protein synthesis inhibitor cycloheximide at 36 μM final concentration; then samples were withdrawn at different time points and analyzed by immunoblotting.
4
Cell Cycle Analysis of DHEA-treated Hepatocytes
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Hepatocytes were plated in 6-well plates (2 × 10 6 cells per well) and treated with 0, 0.1, 1 or 10 μM DHEA for 24 h. After incubation, the cells were harvested and fixed in 1 ml 75 % cold ethanol, and then incubated at -20 °C for 18 h. The cells were centrifuged at 1,000 rpm for 5 min, and the cell pellets were resuspended in 500 μl propidium iodine (50 μg/ml) containing 5 U RNase and incubated on ice for 30 min. Cell cycle distribution was calculated from 10,000 cells with ModFit LTTM software (Becton Dickinson, San Jose, CA, USA) using FACScaliber (Becton Dickinson, San Jose, CA, USA).
5
Cell Cycle Analysis of MDA-MB231 Cells
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Cell cycle distribution was analyzed using flow cytometry as described previously (20) . Briefly, MDA-MB231 cells, seeded in 6-well culture plates, were treated for 24 h with test compounds. Thereafter, cells were trypsinized, harvested and fixed in 1 ml 70 % cold ethanol in test tubes and incubated at -20 for 30 min. After incubation, cells were centrifuged at 1,500 rpm for 5 min and cell pellets were re-suspended in 500 μl PI (10 μg/ml) containing 300 μg/ml RNase (Sigma-Aldrich, St. Louis, MO, USA). Then cells were incubated on ice for 30 min and filtered through a 53-μm nylon mesh. Cell cycle distribution was calculated from 10,000 cells with ModFit LTTM software, Becton Dickinson (San Jose, CA, USA) using FACScaliber (Becton Dickinson).
6
Cell Cycle Analysis by Flow Cytometry
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L02 and HepG2 cells were cultivated in 6-well plates treated by the above methods and then cultured for 48 hrs. The cells were collected, fixed with 70% ethanol overnight and then resuspended in cold PBS, and then incubated with 1 mg/mL 7-AAD (420404; BioLegend, CA, USA) at 37°C for 15 min. The samples were detected by using FACScalibur flow cytometer (BD Biosciences, San Diego, CA, USA), and the proportions of cells in the G1, S, and G2 phases were investigated by using ModFit LTTM software (BD Biosciences, San Diego, USA).
7
Cell Cycle Analysis Using Propidium Iodide
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Cell Cycle and Apoptosis Analysis Kit (Beyotime, Shanghai, China) was applied for cell cycle analysis. Briefly, the cells were transfected with siRNAs (Mock-siRNA, siL21-1 and siL21-2) at a concentration of 40 nM for 72 h, and then were harvested and fixed with cold 75% ethanol at 4°C overnight. The fixed cells were collected and suspended in phosphate-buffered saline (PBS) buffer containing 10 μg/ml propidium iodide (PI) and 10 μg/ml RNase A, and then incubated for 30 min at room temperature. DNA content was measured by the BD FACSCalibur (BD Biosciences, San Jose, CA, United States), and each histogram was constructed with the data from at 10,000 events. The data were analyzed and expressed as percentages of total gated cells using the Modfit LTTM Software (BD Biosciences, San Jose, CA, United States).
8
Cell Cycle and Superoxide Analysis
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EdU incorporation during DNA synthesis was evaluated using the Click-it EdU flow cytometer assay according to manufacturer protocol. 10000 events were acquired using BD Accuri C6 Flow Cytometer and cell cycle was analyzed using BD Accuri C6 Software (BD Biosciences).
Tet-ON Tap63γ myoblasts were grown in presence or absence of doxycyclin (2µg/µl) for 8, 16 and 24 hours, fixed 30 minutes at 4 °C in methanol:aceton (4:1), incubated 20 minutes at 37̊C with RNAse A (20µg/ml) (Sigma, USA) and 20 minutes at room temperature with PI (50µg/ml) (Sigma, USA). 10000 events were acquired using BD FacsCalibur, cell cycle phases and subG1 populations were evaluated by ModFit LTTM software (BD Biosciences).
For the detection of mitochondrial anion superoxide, Scr, sh1-p63 and sh2-p63 cells were incubated with MitoSox Red dye (5µM, Invitrogen) for 10 minutes at 37°C and 10000 events were acquired using BD Accuri C6 Flow Cytometer and analyzed using BD Accuri C6 Software (BD Biosciences).
Tet-ON Tap63γ myoblasts were grown in presence or absence of doxycyclin (2µg/µl) for 8, 16 and 24 hours, fixed 30 minutes at 4 °C in methanol:aceton (4:1), incubated 20 minutes at 37̊C with RNAse A (20µg/ml) (Sigma, USA) and 20 minutes at room temperature with PI (50µg/ml) (Sigma, USA). 10000 events were acquired using BD FacsCalibur, cell cycle phases and subG1 populations were evaluated by ModFit LTTM software (BD Biosciences).
For the detection of mitochondrial anion superoxide, Scr, sh1-p63 and sh2-p63 cells were incubated with MitoSox Red dye (5µM, Invitrogen) for 10 minutes at 37°C and 10000 events were acquired using BD Accuri C6 Flow Cytometer and analyzed using BD Accuri C6 Software (BD Biosciences).
9
Cell cycle analysis by PI staining
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Cell cycle analysis was performed using the propidium iodide (PI)-staining method as described previously [36 (link)]. Briefly, cells were treated with decitabine at a concentration of 500 nM for 72 h and then harvested and fixed in cold 75% ethanol at 4 °C overnight. The fixed cells were collected, suspended in phosphate-buffered saline (PBS) buffer containing 10 μg/mL PI and 10 μg/mL RNase A, and incubated for 30 min at room temperature. DNA content was measured using a BD FACSCalibur (BD Biosciences, San Jose, CA, USA), and each histogram was constructed from data of at least 20,000 events. The data are expressed as percentages of total gated cells using Modfit LTTM Software (BD Biosciences).
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